CN105779572A - Chip and method for capturing target sequences of tumor susceptibility genes, and mutation detection method - Google Patents
Chip and method for capturing target sequences of tumor susceptibility genes, and mutation detection method Download PDFInfo
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Abstract
The invention discloses a chip and method for capturing target sequences of tumor susceptibility genes, and a mutation detection method. The chip is a liquid chip which binds with probe groups capable of simultaneously capturing at least 5, preferably at least at least 10, preferably at least at least 20, preferably at least at least 30, preferably at least at least 50, preferably at least at least 80, preferably at least at least 100, preferably at least at least a10, or preferably all of 115 genetic tumor susceptibility genes as shown in a table 1. The mutation detection method comprises a step of capturing the target sequences of the genetic tumor susceptibility genes by using the liquid chip and a step of carrying out sequencing by using G2 high-flux sequencing technology so as to find out mutation sites. The mutation detection method has the advantages of a wide application scope, high efficiency, comprehensiveness and easy operation, can detect replacement of a single base, insertion or deletion of a single base/multiple bases and deletion/amplification of large fragments and is capable of realizing high-efficiency comprehensive detection of common tumor susceptibility gene mutation.
Description
Technical field
The present invention relates to chip and sequencing technologies field, particularly relate to tumor susceptibility gene target sequence and catch chip, method and mutation detection methods.
Background technology
In recent years, the M & M of China's malignant tumor (i.e. cancer) constantly raises, and malignant tumor has become the public health problem that China is great.The morbidity mode of tumor can be divided into heritability, familial and sporadic three types, and wherein the sickness rate of hereditary tumor accounts for the 5%-10% of tumor total incidence.Hereditary tumor comes from some specific gene and there occurs germ line mutation, and this sudden change can be hereditary in family, causes that offspring suffers from cancer risk and increases, and those easily occur the gene of germ line mutation to be referred to as hereditary tumor tumor susceptibility gene.Carry the individuality of hereditary tumor tumor susceptibility gene, the Hazard ratio ordinary people that its cancer occurs exceeds several times even decades of times, age of onset is generally also Zao than distributing type simultaneously, also points out its family member to have the risk that high hereditary tumor tumor susceptibility gene carries simultaneously.Such as, its probability suffered from breast cancer throughout one's life of the BRCA gene germ line mutation carrier in women population is up to 50%-80%, and the cumulative lifetime risk of population is then less than 7%.Additionally, about 25% colorectal cancer has genetic background, if hereditary nonpolyposis colorectal cancer is to be occurred pathogenic mutation to cause by its tumor susceptibility gene mismatch repair gene (MLH1, MUTYH, MSH2 etc.), the carriers of mutation accumulative risk suffering from colorectal cancer in all one's life is about 80%, and the risk of population is then about 2%.For breast carcinoma, colorectal cancer etc., these have the cancer of heritability tendency, American National comprehensive cancer net (NCCN) gives clear and definite risk assessment and provisional monitor instructs, require to meet hereditism to enter to organize the crowd of standard and carry out relevant susceptibility gene mutation check and evaluation, and select suitable provisional monitor measure according to assessment result, effectively reduce sickness rate, delay tumor invasion, to improve the quality of living.Therefore, the early prediction of the detection of hereditary tumor susceptibility gene mutation and onset risk has important practical value and meaning in treatment and prevention of tumour works.
Tumor susceptibility gene mutation detection methods conventional at present has multiplex ligation-dependent probe amplification (Multiplexligation-dependentProbeamplification, MLPA), PCR-denaturing high-performance chromatography (DHPLC) technology and PCR-conventional sequencing technology etc..It addition, developing rapidly and universal along with second filial generation high-flux sequence (NextGenerationSequencing, NGS) technology, the tumor susceptibility gene abrupt climatic change based on NGS is tentatively developed and applies.Each technology introduction and technical characterstic are as follows:
1. multiplex ligation-dependent probe amplification (MLPA) technology
MLPA technology is the earliest by Holland scholar Dr.SchoutenJP (SchoutenJP, McElgunnCJ, WaaijerR, etal.Relativequantificationof40nucleicacidsequencesbymul tiplexligation-dependentprobeamplification.NucleicAcidsR es.2002Jun15；30 (12): e57) proposed in 2002 years, it is a kind of high flux, the technology carrying out qualitative and quantitative analysis for target sequence in determined nucleic acid, it utilizes simple heterozygosis to connect and pcr amplification reaction, can detect the copy number change of 40 different nucleotide sequences in single reaction pipe simultaneously.Up to the present multiple field such as gene test and gene diagnosis it is widely used in, as numerical abnormalities of chromosomes, hereditary gene delection repeat (such as Erb's atrophy, hereditary nonpolyposis colorectal cancer etc.), gene methylation detection etc..Detection specificity is high, precision is high for it, repeatability is strong, easy and simple to handle, it is suitable for high throughput testing, but this technology also has its limitation, individual cells sample can't be detected at present, can not be used for detecting short tandem repeat polymorphism (STR) and chromosomal balanced translocation, point mutation for gene internal still can not detect (HuangCH, ChangYY, ChenCH, etal.Copynumberanalysisofsurvivalmotorneurongenesbymulti plexligation-dependentprobeamplification.GenetMed.2007Ap r；9 (4): 241-8) etc., thus be of limited application on tumor susceptibility gene abrupt climatic change.
2.PCR-denaturing high-performance chromatography (DHPLC) technology
PCR-DHPLC technology is used to a kind of technology of detection single nucleotide polymorphism (SNP) and base mutation at first, analyzes system also referred to as WAVE nucleotide fragments.It utilizes the principle of DHPLC, and the DNA fragmentation after pcr amplification and buffer (TEAA) are mixed to form mobile phase, and mobile phase is by high drive, by a DNASep detached dowel, DNA fragmentation can be easily separated and analyze.Denaturation temperature is to affect the key factor that DNA fragmentation is analyzed, and denaturation temperature raises, and retention time shortens.Owing to the feature of unwinding of heteroduplex (mispairing) DNA and homoduplex DNA is different, when identical partial denaturation, heteroduplex is because having the existence in mispairing district and more changeableness, it is shorter than homoduplex by chromatographic column retention time, therefore be first eluted, chromatogram shows as bimodal or multimodal elution curve, so that the two is distinguished.
PCR-DHPLC method does not need the loaded down with trivial details operation (KodamaCS such as encapsulating, loading, electrophoresis, Cuadros-OrellanaS, BandeiraCH, etal.UseofPCR-DHPLCwithfluorescencedetectionforthecharac terizationofthebacterialdiversityduringcassava (ManihotesculentaCrantz) fermentation.GenetMolRes.2014Feb28；13(1):1304-13；SoumittraN, MeenakumariB, ParijaT, etal.Moleculargeneticsanalysisofhereditarybreastandovari ancancerpatientsinIndia [J] .HeredCancerClinPract, 2009,7 (1): 13), fast and automatically, detection lug segment length can reach 1500bp, rate of accuracy reached more than 96%.But its detection mutation type is limited, and need the special primer of design to carry out pcr amplification for each mutational site, sample makes consumption count with detecting position to be multiplied, therefore in the face of the multidigit point detection demand of tumor susceptibility gene, can only separate and repeatedly carry out, take time and effort, it is difficult to scale, cost degradation (Cai Zhen, Zheng Lei. many target genes Parallel detection provides new model [J] for tumor individual therapy. molecular diagnosis and treatment magazine, 2013 (6): 361～366).
3.PCR-conventional sequencing technology
PCR primer direct Sequencing technology is an important technology in molecular biology and genomics research, is widely used in detection in Gene Mutation, hereditary diagnosis, single nucleotide polymorphism research, genome overlapping sequence group etc..Compared with tradition cloning and sequencing technology, directly the DNA of pcr amplification is checked order, eliminate cloning process consuming time, it is to avoid the repetitive operations such as traditional antibacterial culturing, template extraction, it is possible to from a small amount of primary sample, obtain correct DNA sequence information.Although PCR primer direct Sequencing technology has advantage quick, easy, that stabilize the economy, but it there is also not foot point, as low in flux, cost is high, operating procedure is relatively complicated, automaticity is low, it is difficult to large-scale application detects in the multidigit point of tumor susceptibility gene.
4. based on the mutation detection techniques of NGS
DNA sequencing technology starts from the seventies in last century, the variations such as the nucleic acid mutation of gene, insertion and rearrangement can be told more intuitively due to sequencing technologies, the nucleotide sequence of detection target gene has the advantage of uniqueness, is widely used in the field such as selection and molecule parting of tumor-targeting drug.Along with the development of NGS sequencing technologies, order-checking time and spend relatively first generation Sanger sequencing technologies to be substantially reduced, and the flux checked order and the degree of depth are greatly improved.Full transcript profile and genome sequencing technology combine, and can not only provide complete individual cells genetic transcription collection, moreover it is possible to detect novel gene sudden change, transcribe, gene fusion etc. abnormal, even help to explain genomic function.
Summary of the invention
The present invention provides a kind of tumor susceptibility gene target sequence to catch chip, method and mutation detection methods, there is advantage applied widely, efficient, comprehensive, easy-operating, can detect the single base replacement in target sequence, single base/many bases are inserted or are lacked and large fragment deletion/amplification, it is possible to meet efficient, the complete detection of kinds of tumor susceptibility gene mutation.
According to the first aspect of the invention, the present invention provides a kind of hereditary tumor tumor susceptibility gene target sequence to catch chip, described chip is liquid-phase chip, it is combined with can catch in the hereditary tumor tumor susceptibility gene of 115 shown in table a kind simultaneously at least 5 kinds, preferably at least 10 kinds, preferably at least 20 kinds, preferably at least 30 kinds, preferably at least 50 kinds, preferably at least 80 kinds, preferably at least 100 kinds, preferably at least 110 kinds, preferably all of target acquistion region probe combinations.
As the preferred version of the present invention, described target acquistion region includes all exon regions and exon and intron join domain.
As the preferred version of the present invention, the liquid-phase chip that described liquid-phase chip is is carrier with the NimblegenEZ chip of Roche.
The hereditary tumor that the present invention is suitable for is breast carcinoma, ovarian cancer, intestinal cancer, gastric cancer, carcinoma of prostate, carcinoma of endometrium, leukemia, medulloblastoma, ganglioneuroblastoma, neuroblastoma, Multiple Endocrine tumor, multiple neurofibromatosis, pulmonary carcinoma, pulmonary blastoma, pheochromocytoma, osteosarcoma, melanoma, rhabdomyosarcoma, basal cell tumor, parathyroid carcinoma, thyroid carcinoma, lymphoma, endocrine tumor, skin carcinoma, smooth cell tumor, renal carcinoma, nephroblastoma, adrenocortical carcinoma, meningioma, bladder cancer, retinocytoma, chromaffin cell tumor, glioma, exophytic osteosarcoma, gastrointestinal stromal tumor, thrombocytosis, one or more in cylindroma and cancer of pancreas.
According to the second aspect of the invention, the present invention provides a kind of hereditary tumor tumor susceptibility gene target sequence catching method, catches, including the hereditary tumor tumor susceptibility gene target sequence used described in first aspect, the step that chip and DNA sample to be captured carry out hybridizing.
According to the third aspect of the invention we, the present invention provides the detection method of the gene mutation of a kind of hereditary tumor tumor susceptibility gene, catches, including using the hereditary tumor tumor susceptibility gene target sequence described in first aspect, the step that chip and DNA sample to be captured carry out hybridizing；With use second filial generation high throughput sequencing technologies to catching the step that the target dna obtained checks order.
As the preferred version of the present invention, the detection method of the gene mutation of described hereditary tumor tumor susceptibility gene comprises the steps:
(1) genomic DNA sample to be detected is broken into fragment, it is preferable that be broken into the fragment that length is 220-400bp；
(2) fragment that step (1) is interrupted is purified, end reparation, adds joint, and expands with PCR；
(3) product step (2) obtained and the hereditary tumor tumor susceptibility gene target sequence described in first aspect are caught chip and are hybridized, and capture the DNA fragmentation in described target acquistion region；
(4) DNA fragmentation in described target acquistion region step (3) captured elutes, it is thus achieved that the target dna of needs；
(5) target dna that step (4) obtains is used to build sequencing library；
(6) use the sequencing library that step (5) is obtained by second filial generation high-flux sequence to check order, obtain reading section (reads)；
(7) reads step (6) obtained compares with reference to genome.
As the preferred version of the present invention, the form of described gene mutation is that base is replaced, inserted or disappearance and fragment deletion or amplification.The method of the present invention is applicable to single base replacement, single base/many bases are inserted or the polytype such as disappearance and large fragment deletion/amplification, and compared to existing technology, the method scope of application of the present invention is wider.
As the preferred version of the present invention, the joint in described step (2) is that CG (CompleteGenomics) checks order the A joint of platform；Double-stranded DNA, particularly as follows: again carry out PCR, is carried out cyclisation by described (5), carries out enzyme action at 26bp place, described A joint two ends, otch is carried out end reparation, adds B joint, then DNA double chain is separated into strand, and described strand is carried out cyclisation.
In one embodiment of the present of invention, use CG order-checking platform to carry out second filial generation high-flux sequence, the structure of corresponding sequencing library, adopt the sequencing library construction method that this platform is general.The method uses the cutting characteristic of III class restriction endonuclease, the genomic DNA at 26bp place, enzyme action A joint both sides, then otch is carried out end reparation, add B joint, then DNA double chain is separated into strand, and described strand is carried out cyclisation.Wherein, A joint and B joint are the universal joints of this platform.
Preferred version as the present invention, second filial generation high-flux sequence in described step (6) is CG order-checking, and ensureing that the original data volume of each described sequencing library reaches more than 0.6Gb, the order-checking degree of depth of target area reaches 400 × more than, target area coverage reaches more than 99%.
As the preferred version of the present invention, described step (7) particularly as follows:
First, reads comparison order-checking obtained, to reference genome, is not allow for inserting and disappearance, it is preferable that compare with Teramap；
Then, identify and be likely to and described with reference to the different region of genome, it would be possible to comparison is picked out to the reads in these regions and carried out local and assemble, and compares assembling the sequence obtained with described reference genome, it is determined that various types of sudden changes；
Thereafter, the sudden change detected is given a mark, it is preferable that give a mark with varScoreVAF and varScoreEAF；
Finally, filtering out lower than predetermined quality, lower than desired depth, preferred alt_depth < 2/allreads < 5, lower than the sudden change of preset frequency, preferred MAF < 0.25, finally suddenlyd change list.
According to the fourth aspect of the invention, the present invention provides the gene mutation of the hereditary tumor tumor susceptibility gene that the detection method of the gene mutation of the hereditary tumor tumor susceptibility gene using hereditary tumor tumor susceptibility gene target sequence described in first aspect to catch described in chip or the third aspect detects, described gene mutation is that the coded sequence shown in SEQIDNO:1 lacks the 640th nucleotide A base；Or the coded sequence shown in SEQIDNO:2 is inserted with base A between the 5800th and 5801 nucleotide, or between the 274th and 275 nucleotide, it is inserted with base A, or at the 1219th nucleotide position generation nonsense mutation；Or the coded sequence shown in SEQIDNO:3 lacks at the 1709th to 1710 nucleotide generation base AG.
Said gene sudden change can indicate as the hereditism of hereditary breast cancer, when detecting that said gene sudden change illustrates that corresponding individuality would be likely to occur higher hereditary breast cancer occurrence risk.Therefore, said gene sudden change can be used in gene test, or for preparing in the reagent detecting hereditary breast cancer.
The present invention is by catching associating second filial generation high throughput sequencing technologies to common inherited tumor susceptibility gene target area, and in conjunction with analysis of biological information method, realize the complete detection in hereditary tumor susceptibility gene mutation site, there is the technical advantages such as detection flux is high, highly sensitive, high specificity, accuracy high, coverage is wide, effectively solve the problems such as hereditary tumor susceptibility gene mutation region is wide, mutational site is uncertain.Concrete advantage illustrates as follows:
1. detection range is wide: the present invention can realize detection simultaneously and include the some or all variation information in 115 tumor susceptibility genes that the cancers such as breast carcinoma/ovarian cancer, colorectal cancer, cancer of pancreas, gastric cancer, retinoblastoma are relevant, and various mutations type can be detected, for instance SNP, insertion and deletion (InDel) etc..
2. flux is high: use liquid-phase chip can catch the DNA of multiple sample simultaneously；And, multiple samples can be carried out the order-checking (once go up machine order-checking and can obtain the data volume of about 2T, target area mean coverage can reach more than 99%) of high flux, high depth, high coverage by second filial generation high-flux sequence platform (such as CG order-checking platform) simultaneously；Plus high performance computer information processing ability, it is possible to realize the variation detection of great amount of samples and analyze simultaneously.
3. high sensitivity, specificity, accuracy: based on second filial generation high-flux sequence platform (as CG check order platform) high throughput sequencing technologies, DNA sequence information accurately can be obtained, use the analysis method of bioinformatics, almost all of sudden change in target zone can be detected, and resolution can reach single base level.
4. it is convenient to implement: current many chip production business can provide the synthesis of unrelated probe, possesses the feature of economical and efficient, is commercially available at any time, simultaneously according to gene order-checking platform, using the teaching of the invention it is possible to provide high-throughout order-checking service, meets coherent detection demand.And be analyzed by related software, it is easy to accomplish automatization.
Accompanying drawing explanation
Fig. 1 is the sequencing data abrupt climatic change flow process in the present invention；
Fig. 2 is the agarose gel electrophoresis testing result of the genomic DNA extracted from 34 example patient with breast cancer's blood samples in the embodiment of the present invention, and wherein M represents that DNAMarkerDL2000,1-34 represent 34 example genomic DNAs respectively；
The Sanger order-checking gained peak figure of No. 5, No. 10, No. 15, No. 25 and No. 31 samples in Fig. 3 to Fig. 7 respectively embodiment of the present invention.
Detailed description of the invention
The method catching associating high-flux sequence for common inherited tumor susceptibility gene target sequence set forth in the present invention is based on the pathogenetic background of hereditary tumor and the demand of susceptibility gene mutation detection technique thereof and designs.nullThe present invention is with all exon regions of common inherited tumor susceptibility gene (115 shown in table a kind gene) and exon and intron join domain for target acquistion region，It is designed to catch the probe combinations in all target sequence regions simultaneously，Customization liquid-phase chip (is produced by Roche Holding Ag，NimblegenEZ chip)，And combine CompleteGenomics (CG) second filial generation high throughput sequencing technologies and information analysis techniques，All target sequences captured are checked order and different types of abrupt information analysis，To understand, whether target sample exists the germ line mutation that can result in the tumor susceptibility gene that tumor invasion risk raises，And tumor prevention and monitoring is instructed according to emergent properties，Hereditary tumor susceptibility gene mutation data can be accumulated rapidly simultaneously，It is provided with force data support for industrialization.This invention has the advantages such as applied widely, efficient, comprehensive, easy operation, single base in detection target sequence is replaced simultaneously, single base/many bases are inserted or the mutation type such as disappearance and large fragment deletion/amplification, meet efficient, the complete detection of kinds of tumor susceptibility gene mutation.
Method for designing and the experimentation of one embodiment of the present of invention are as follows:
1. investigation and analysis, it is determined that target gene
Collect the tumor susceptibility gene relevant to tumor invasion, finally determine 115 hereditary tumor tumor susceptibility genes.Table 1 and table 2 list the title of the hereditary tumor corresponding to the title of each tumor susceptibility gene and tumor susceptibility gene respectively, and wherein different tumors may exist identical tumor susceptibility gene.
1.115 hereditary tumor tumor susceptibility gene lists of table
Hereditary tumor title corresponding to each tumor susceptibility gene of table 2
2. determine the target acquistion region of each target tumor susceptibility gene, customize liquid-phase chip
With all exon 1s of 115 target tumor susceptibility genes and exon and intron join domain for general objective region, customization liquid phase catches chip, specifically by Roche Holding Ag according to the target acquistion region design capture probe provided, produces into NimblegenEZ chip.The final target area probe sequence obtained comprises 23434 probes, the length of every probe sequence is 121bp, respectively comprising the sequence label of 16bp and 15bp before and after sequence, it is GAAGCGAGGATCAACT (SEQIDNO:4) and CATTGCGTGAACCGA (SEQIDNO:5) respectively that the sequence set of former and later two sequence labels becomes.The two sequence label respectively restriction enzyme site and transcription site, two ends are all used to design PCR primer, and transcription site is used for transcribing simultaneously, plays the effect being transcribed into rna probe.
3. extract sample DNA
Use DNA extraction kit (QIAGENDNABloodminikit) to extract genomic DNA from person under inspection's blood, after being used for, continue storehouse experiment.
4. the library construction that target area is caught
First, with interrupting instrument, genomic DNA is broken at random 220-400bp small fragment, DNA is purified, end reparation, adds the A joint joint of platform (CG check order) and expand with PCR；Then, by base pair complementarity principle, use the DNA that liquid phase catches the genome target region that the capture probe pair on chip matches with it to hybridize, capture the DNA of target area, by elution reagent, target dna is eluted from probe, it is thus achieved that the target dna of needs；Thereafter, again carry out PCR, double-stranded DNA is carried out cyclisation, carry out enzyme action at 26bp place, A joint two ends, otch is carried out end reparation, add B joint (joint of CG order-checking platform)；Finally, separate DNA double chain and become strand, strand is carried out cyclisation, is namely built into the library that target area is caught.
5. library Quality Control
Using Qubit and electrophoresis detection sample strip and degraded situation, use PAGE-Urea to measure Insert Fragment size cases, measure the molar concentration in library with BMG, only sample library is qualified could go up machine order-checking.
6. go up machine order-checking
Catch library quality inspection qualified after, illustrate to carry out upper machine order-checking according to the CG platform operations that checks order, and ensure that the original data volume in each library reaches more than 0.6Gb, the target area order-checking degree of depth reaches 400 × more than, target area coverage reaches more than 99%.
7. sequencing data filtration, comparison, mutation analysis
After having checked order, lower machine data are carried out analysis of biological information, flow process following (as shown in Figure 1): first, reads Teramap software (offer of CompleteGenomics company) comparison order-checking obtained is to reference to, on genome Hg19 (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/), being not allow for inserting and disappearance；Then, identify and be likely to region different with reference genome, it would be possible to comparison is picked out to the reads in these regions and carried out local and assemble, and will assemble the sequence and reference genome comparison that obtain, it is determined that various types of variations；Thereafter, with varScoreVAF (offer of CompleteGenomics company) and varScoreEAF (offer of CompleteGenomics company), the sudden change detected is given a mark；Finally, filter out the sudden change of low quality (mark VQLOW), low depth (supporting that the reads of sudden change is less than 2), low frequency (sudden change allele frequency (MAF) is less than 0.25), obtain last sudden change list.
8. accidental data is understood, it is thus achieved that the mutational site relevant to hereditary tumor morbidity
For the sudden change detected, first with annovar (http://www.openbioinformatics.org/annovar/) annotation, obtain the information of their gene information, gene region information, transcript information, aminoacid change；nullThen public database dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) is used,1000genome(http://www.1000genomes.org/)，hapmap(http://hapmap.ncbi.nlm.nih.gov/),BIC(http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/)，HGMD(http://www.hgmd.cf.ac.uk/ac/index.php)，The information of software SIFT (http://sift.jcvi.org/) and bibliographical information understands these sudden changes，Determine that whether they are relevant to hereditary tumor morbidity.
The present invention is by catching associating second filial generation high throughput sequencing technologies to common inherited tumor susceptibility gene target area, and in conjunction with analysis of biological information method, realize the complete detection in the common inherited tumor susceptibility gene mutational site such as hereditary breast cancer, colorectal cancer, there is the technical advantages such as detection flux is high, highly sensitive, high specificity, accuracy high, coverage is wide, effectively solve the problems such as hereditary tumor susceptibility gene mutation region is wide, mutational site is uncertain.
Below in conjunction with embodiment, embodiment of the present invention are described in detail.Embodiment is only used for further illustrating the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete technology or condition person in embodiment, technology or condition described by the document in this area or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be and can pass through the conventional products that city's purchase obtains.
Embodiment: catch the hereditary breast cancer susceptibility gene mutation detection of associating CompleteGenomics order-checking based on chip
1. experiment purpose
The present embodiment detection is analyzed 34 examples and is had SNP and the Indel catastrophe of hereditary breast cancer tumor susceptibility gene in patient with breast cancer (proband) blood DNA of genetic predisposition, confirms whether this lot sample originally exists the pathogenic mutation of the relevant tumor susceptibility gene of breast carcinoma.
2. experiment material
Sample information: 34 examples meet hereditary breast cancer and enter to organize patient with breast cancer's blood sample of standard, detailed sample enters group standard for (following condition meets one): (1) breast carcinoma ill age≤40 years old；(2) male breast carcinoma patient, age of onset is not limit；(3) three Breast Cancer Patients with Negative Axillaries, age of onset is not limit；(4) BILATERAL BREAST CANCER patient (can simultaneously occur, it is possible to occur time different), age of onset is not limit；(5) there is susceptibility gene mutation in any patient with breast cancer and family；(6) I suffered from breast cancer at any age, and: 1) >=1 close relative suffer from breast cancer and ill age≤50 years old, or 2) >=2 close relatives (need to from same Fan family race: maternal or paternal) suffer from breast cancer or cancer of pancreas, age of onset is not limit, or 3) >=1 close relative has ovarian cancer (including carcinoma of fallopian tube and Primary peritoneal carcinoma), age of onset is not limit, or 4) >=1 paternal line or maternal family member suffer from breast carcinoma and >=1 paternal line or maternal family member suffers from following malignant tumor (particularly age of onset≤50 year old): cancer of pancreas, carcinoma of prostate, sarcoma, adrenocortical carcinoma, cerebroma, carcinoma of endometrium, leukemia/lymphoma, thyroid carcinoma, digestive tract polyposis (hamartoma type), diffusivity gastric cancer.
Key instrument: pipettor, PCR instrument, centrifuge, turbula shaker, electrophresis apparatus,DNA interrupts instrument, magnetic frame, dry type constant-temperature metal bath, CompleteGenomics sequenator.
Main agents: QIAGEN blood DNA extracts test kit, and storehouse reagent built by CompleteGenomics platform, and human genome (hg19) region of interest within is about the liquid phase probe (Roche Holding Ag, NimblegenEZ chip) of 600Kb.
3. experimental technique and step
(1) poba gene group DNA extraction
Use QIAGENDNABloodminikit, and according to the extraction description of this test kit, from patient with breast cancer's blood sample, extract genomic DNA, useDetection DNA concentration, in principle the DNA acquisition amount >=2 μ g of every part of sample, then whether electrophoresis detection DNA complete and palliating degradation degree, and deposition condition is: the agarose gel of 1%, electrophoretic voltage 4V/cm, electrophoresis time 45min.Genome dna electrophoresis testing result is as in figure 2 it is shown, result shows: DNA is complete, is substantially free of degraded.
(2) library construction before order-checking
Building flow process with reference to CompleteGenomics exon group sequencing library, concretely comprise the following steps: 1) genomic DNA interrupts, is broken into 220-400bp small fragment at random, is then purified and end reparation；2) adding A joint, performing PCR of going forward side by side expands；3) by the principle of base pair complementarity, the DNA of the genome target region that use probe pair matches with it is hybridized, and captures the DNA of target area, is eluted by target dna by elution reagent from probe；4) pcr amplification, carries out double-strand cyclisation to DNA；5) at 26bp place, joint two ends enzyme action；6) end reparation, adds B joint；7) separate DNA double chain and become strand, strand is carried out cyclisation；8) single-stranded loop chemoattractant molecule rolling-circle replication, forms DNA nanosphere (DNB).
(3) CompleteGenomics high-flux sequence
To Quality Control library DNA after qualified, carry out upper machine order-checking according to the operating instruction of CompleteGenomics order-checking.The order-checking original data volume of each sample obtained reaches more than 0.6Gb, and the average order-checking degree of depth of target area reaches 400 ×, target area coverage is more than 99%.The sequencing data quality condition of 34 example samples is as shown in table 3.
Table 334 example patient with breast cancer's sample object region high-flux sequence quality of data situation
(4) sequencing data processes and analysis of biological information
According to the order-checking data processing spec of platform and actual demand, lower machine the data obtained is carried out preliminary treatment and carries out information analysis.First, carrying out sequence alignment, sequence Teramap software comparison order-checking obtained, to the reference genome (hg19) of people, is not allow for inserting and disappearance simultaneously；Then, identify and be likely to region different with reference genome, it would be possible to comparison is picked out to the sequence in these regions and carried out local and assemble, and will assemble the sequence and reference genome comparison that obtain, it is determined that various types of sudden changes；Hereafter, use varScoreVAF and varScoreEAF that the sudden change detected is given a mark；Finally, filter out low quality (VQLOW), the sudden change of low depth (alt_depth < 2/allreads < 5) and low frequency (MAF < 0.25), obtain last mutational site information.
(5) accidental data of breast cancer susceptibility gene is understood, find out pathogenic mutation
SNP and the Indel accidental data list obtained through analysis of biological information needs to carry out further understanding, screening to it, understands step as follows: first, screened by SNP and the Indel accidental data of all breast cancer susceptibility genes；Secondly, the exon region of breast cancer susceptibility gene and exon and the splice mutation of intron join domain, nonsense mutation and frameshift mutation are screened；Then, at BIC (BreastCancerInformationCore, http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/) data base, HGMD (TheHumanGeneMutationDatabase, http://www.hgmd.cf.ac.uk/ac/index.php) mutational site screened is scanned for by data base and research papers, whether specify mutational site is known sudden change, if not searching corresponding report in existing database or Research Literature, then illustrate that this sports new mutation.
4. experimental result
By the sequencing data of 34 example samples is analyzed, data base is to when understanding, find that 5 example proband are respectively present the mutational site of the relevant tumor susceptibility gene of breast carcinoma altogether, find that these mutational sites were not all reported the data base such as Research Literature, BIC at present simultaneously, therefore be judged to new mutational site.All the other 29 example samples do not find the pathogenic mutation of related gene, only exist benign polymorphic variation (i.e. single nucleotide polymorphism).In this lot sample basis, the mutational site by analyzing after understanding is as shown in table 4.
Table 4 checks order 5 mutational site information of gained from 34 example proband's blood DNA samples
The following is the detailed description to 5 the new hereditary breast cancer mutational sites found:
(1) wild-type sequence of BARD1 gene coding region is:
nullAtgccggataatcggcagccgaggaaccggcagccgaggatccgctccgggaacgagcctcgttccgcgcccgccatggaaccggatggtcgcggtgcctgggcccacagtcgcgccgcgctcgaccgcctggagaagctgctgcgctgctcgcgttgtactaacattctgagagagcctgtgtgtttaggaggatgtgagcacatcttctgtagtaattgtgtaagtgactgcattggaactggatgtccagtgtgttacaccccggcctggatacaagacttgaagataaatagacaactggacagcatgattcaactttgtagtaagcttcgaaatttgctacatgacaatgagctgtcagatttgaaagaagataaacctaggaaaagtttgtttaatgatgcaggaaacaagaagaattcaattaaaatgtggtttagccctcgaagtaagaaagtcagatatgttgtgagtaaagcttcagtgcaaacccagcctgcaataaaaaaagatgcaagtgctcagcaagactcatatgaatttgtttccccaagtcctcctgcagatgtttctgagagggctaaaaaggcttctgcaagatctggaaaaaagcaaaaaaagaaaactttagctgaaatcaaccaaaaatggaatttagaggcagaaaaagaagatggtgaatttgactccaaagaggaatctaagcaaaagctggtatccttctgtagccaaccatctgttatctccagtcctcagataaatggtgaaatagacttactagcaagtggctccttgacagaatctgaatgttttggaagtttaactgaagtctctttaccattggctgagcaaatagagtctccagacactaagagcaggaatgaagtagtgactcctgagaaggtctgcaaaaattatcttacatctaagaaatctttgccattagaaaataatggaaaacgtggccatcacaatagactttccagtcccatttctaagagatgtagaaccagcattctgagcaccagtggagattttgttaagcaaacggtgccctcagaaaatataccattgcctgaatgttcttcaccaccttcatgcaaacgtaaagttggtggtacatcagggaggaaaaacagtaacatgtccgatgaattcattagtctttcaccaggtacaccaccttctacattaagtagttcaagttacaggcgagtgatgtctagtccctcagcaatgaagctgttgcccaatatggctgtgaaaagaaatcatagaggagagactttgctccatattgcttctattaagggcgacataccttctgttgaataccttttacaaaatggaagtgatccaaatgttaaagaccatgctggatggacaccattgcatgaagcttgcaatcatgggcacctgaaggtagtggaattattgctccagcataaggcattggtgaacaccaccgggtatcaaaatgactcaccacttcacgatgcagccaagaatgggcatgtggatatagtcaagctgttactttcctatggagcctccagaaatgctgttaatatatttggtctgcggcctgtcgattatacagatgatgaaagtatgaaatcgctattgctgctaccagagaagaatgaatcatcctcagctagccactgctcagtaatgaacactgggcagcgtagggatggacctcttgtacttataggcagtgggctgtcttcagaacaacagaaaatgctcagtgagcttgcagtaattcttaaggctaaaaaatatactgagtttgacagtacagtaactcatgttgttgttcctggtgatgcagttcaaagtaccttgaagtgtatgcttgggattctcaatggatgctggattctaaaatttgaatgggtaaaagcatgtctacgaagaaaagtatgtgaacaggaagaaaagtatgaaattcctgaaggtccacgcagaagcaggctcaacagagaacagctgttgccaaagctgtttgatggatgctacttctatttgtggggaaccttcaaacaccatccaaaggacaaccttattaagctcgtcactgcaggtgggggccagatcctcagtagaaagcccaagccagacagtgacgtgactcagaccatcaatacagtcgcataccatgcgagacccgattctgatcagcgcttctgcacacagtatatcatctatgaagatttgtgtaattatcacccagagagggttcggcagggcaaagtctggaaggctccttcgagctggtttatagactgtgtgatgtcctttgagttgcttcctcttgacagctga ( SEQIDNO：1 ).
Wild type BRAD1 gene encodes 778 aminoacid altogether, and the sequencing data of No. 5 proband shows that its 640th nucleotide (A base) in coding region lacks, and frameshift mutation occurs, ultimately results in and can only encode 219 aminoacid.
(2) wild-type sequence of BRCA2 gene coding region is:
nullAtgcctattggatccaaagagaggccaacattttttgaaatttttaagacacgctgcaacaaagcagatttaggaccaataagtcttaattggtttgaagaactttcttcagaagctccaccctataattctgaacctgcagaagaatctgaacataaaaacaacaattacgaaccaaacctatttaaaactccacaaaggaaaccatcttataatcagctggcttcaactccaataatattcaaagagcaagggctgactctgccgctgtaccaatctcctgtaaaagaattagataaattcaaattagacttaggaaggaatgttcccaatagtagacataaaagtcttcgcacagtgaaaactaaaatggatcaagcagatgatgtttcctgtccacttctaaattcttgtcttagtgaaagtcctgttgttctacaatgtacacatgtaacaccacaaagagataagtcagtggtatgtgggagtttgtttcatacaccaaagtttgtgaagggtcgtcagacaccaaaacatatttctgaaagtctaggagctgaggtggatcctgatatgtcttggtcaagttctttagctacaccacccacccttagttctactgtgctcatagtcagaaatgaagaagcatctgaaactgtatttcctcatgatactactgctaatgtgaaaagctatttttccaatcatgatgaaagtctgaagaaaaatgatagatttatcgcttctgtgacagacagtgaaaacacaaatcaaagagaagctgcaagtcatggatttggaaaaacatcagggaattcatttaaagtaaatagctgcaaagaccacattggaaagtcaatgccaaatgtcctagaagatgaagtatatgaaacagttgtagatacctctgaagaagatagtttttcattatgtttttctaaatgtagaacaaaaaatctacaaaaagtaagaactagcaagactaggaaaaaaattttccatgaagcaaacgctgatgaatgtgaaaaatctaaaaaccaagtgaaagaaaaatactcatttgtatctgaagtggaaccaaatgatactgatccattagattcaaatgtagcaaatcagaagccctttgagagtggaagtgacaaaatctccaaggaagttgtaccgtctttggcctgtgaatggtctcaactaaccctttcaggtctaaatggagcccagatggagaaaatacccctattgcatatttcttcatgtgaccaaaatatttcagaaaaagacctattagacacagagaacaaaagaaagaaagattttcttacttcagagaattctttgccacgtatttctagcctaccaaaatcagagaagccattaaatgaggaaacagtggtaaataagagagatgaagagcagcatcttgaatctcatacagactgcattcttgcagtaaagcaggcaatatctggaacttctccagtggcttcttcatttcagggtatcaaaaagtctatattcagaataagagaatcacctaaagagactttcaatgcaagtttttcaggtcatatgactgatccaaactttaaaaaagaaactgaagcctctgaaagtggactggaaatacatactgtttgctcacagaaggaggactccttatgtccaaatttaattgataatggaagctggccagccaccaccacacagaattctgtagctttgaagaatgcaggtttaatatccactttgaaaaagaaaacaaataagtttatttatgctatacatgatgaaacatcttataaaggaaaaaaaataccgaaagaccaaaaatcagaactaattaactgttcagcccagtttgaagcaaatgcttttgaagcaccacttacatttgcaaatgctgattcaggtttattgcattcttctgtgaaaagaagctgttcacagaatgattctgaagaaccaactttgtccttaactagctcttttgggacaattctgaggaaatgttctagaaatgaaacatgttctaataatacagtaatctctcaggatcttgattataaagaagcaaaatgtaataaggaaaaactacagttatttattaccccagaagctgattctctgtcatgcctgcaggaaggacagtgtgaaaatgatccaaaaagcaaaaaagtttcagatataaaagaagaggtcttggctgcagcatgtcacccagtacaacattcaaaagtggaatacagtgatactgactttcaatcccagaaaagtcttttatatgatcatgaaaatgccagcactcttattttaactcctacttccaaggatgttctgtcaaacctagtcatgatttctagaggcaaagaatcatacaaaatgtcagacaagctcaaaggtaacaattatgaatctgatgttgaattaaccaaaaatattcccatggaaaagaatcaagatgtatgtgctttaaatgaaaattataaaaacgttgagctgttgccacctgaaaaatacatgagagtagcatcaccttcaagaaaggtacaattcaaccaaaacacaaatctaagagtaatccaaaaaaatcaagaagaaactacttcaatttcaaaaataactgtcaatccagactctgaagaacttttctcagacaatgagaataattttgtcttccaagtagctaatgaaaggaataatcttgctttaggaaatactaaggaacttcatgaaacagacttgacttgtgtaaacgaacccattttcaagaactctaccatggttttatatggagacacaggtgataaacaagcaacccaagtgtcaattaaaaaagatttggtttatgttcttgcagaggagaacaaaaatagtgtaaagcagcatataaaaatgactctaggtcaagatttaaaatcggacatctccttgaatatagataaaataccagaaaaaaataatgattacatgaacaaatgggcaggactcttaggtccaatttcaaatcacagttttggaggtagcttcagaacagcttcaaataaggaaatcaagctctctgaacataacattaagaagagcaaaatgttcttcaaagatattgaagaacaatatcctactagtttagcttgtgttgaaattgtaaataccttggcattagataatcaaaagaaactgagcaagcctcagtcaattaatactgtatctgcacatttacagagtagtgtagttgtttctgattgtaaaaatagtcatataacccctcagatgttattttccaagcaggattttaattcaaaccataatttaacacctagccaaaaggcagaaattacagaactttctactatattagaagaatcaggaagtcagtttgaatttactcagtttagaaaaccaagctacatattgcagaagagtacatttgaagtgcctgaaaaccagatgactatcttaaagaccacttctgaggaatgcagagatgctgatcttcatgtcataatgaatgccccatcgattggtcaggtagacagcagcaagcaatttgaaggtacagttgaaattaaacggaagtttgctggcctgttgaaaaatgactgtaacaaaagtgcttctggttatttaacagatgaaaatgaagtggggtttaggggcttttattctgctcatggcacaaaactgaatgtttctactgaagctctgcaaaaagctgtgaaactgtttagtgatattgagaatattagtgaggaaacttctgcagaggtacatccaataagtttatcttcaagtaaatgtcatgattctgttgtttcaatgtttaagatagaaaatcataatgataaaactgtaagtgaaaaaaataataaatgccaactgatattacaaaataatattgaaatgactactggcacttttgttgaagaaattactgaaaattacaagagaaatactgaaaatgaagataacaaatatactgctgccagtagaaattctcataacttagaatttgatggcagtgattcaagtaaaaatgatactgtttgtattcataaagatgaaacggacttgctatttactgatcagcacaacatatgtcttaaattatctggccagtttatgaaggagggaaacactcagattaaagaagatttgtcagatttaacttttttggaagttgcgaaagctcaagaagcatgtcatggtaatacttcaaataaagaacagttaactgctactaaaacggagcaaaatataaaagattttgagacttctgatacattttttcagactgcaagtgggaaaaatattagtgtcgccaaagagtcatttaataaaattgtaaatttctttgatcagaaaccagaagaattgcataacttttccttaaattctgaattacattctgacataagaaagaacaaaatggacattctaagttatgaggaaacagacatagttaaacacaaaatactgaaagaaagtgtcccagttggtactggaaatcaactagtgaccttccagggacaacccgaacgtgatgaaaagatcaaagaacctactctattgggttttcatacagctagcgggaaaaaagttaaaattgcaaaggaatctttggacaaagtgaaaaacctttttgatgaaaaagagcaaggtactagtgaaatcaccagttttagccatcaatgggcaaagaccctaaagtacagagaggcctgtaaagaccttgaattagcatgtgagaccattgagatcacagctgccccaaagtgtaaagaaatgcagaattctctcaataatgataaaaaccttgtttctattgagactgtggtgccacctaagctcttaagtgataatttatgtagacaaactgaaaatctcaaaacatcaaaaagtatctttttgaaagttaaagtacatgaaaatgtagaaaaagaaacagcaaaaagtcctgcaacttgttacacaaatcagtccccttattcagtcattgaaaat.
Wild type BRCA2 gene encodes 3418 aminoacid altogether, and the breast cancer susceptibility gene mutation of 10,15 and No. 25 proband's samples occurs on BRCA2 gene coding region, and concrete mutational site is at the 5800th, 274 and 1219 nucleotide respectively.Wherein, the sequencing data of No. 10 proband shows that it inserts base A between the 5800th and 5801 nucleotide, frameshift mutation occurs, ultimately results in and can only encode 1943 aminoacid；The sequencing data of No. 15 proband shows that it inserts base A between the 274th and 275 amino acids, frameshift mutation occurs, ultimately results in and can only encode 99 aminoacid；The sequencing data of No. 25 proband shows that it is at the 1219th nucleotide position generation nonsense mutation, ultimately results in and can only encode 406 aminoacid.
(3) wild-type sequence of PALB2 gene coding region is:
nullAtggacgagcctcccgggaagcccctcagctgtgaggagaaggaaaagttaaaggagaaattagcattcttgaaaagggaatacagcaagacactagcccgccttcagcgtgcccaaagagctgaaaagattaagcattctattaagaaaacagtagaagaacaagattgtttgtctcagcaggatctctcaccgcagctaaaacactcagaacctaaaaataaaatatgtgtttatgacaagttacacatcaaaacccatcttgatgaagaaactggagaaaagacatctatcacacttgatgttgggcctgagtcctttaaccctggagatggcccaggaggattacctatacaaagaacagatgacacccaagaacattttccccacagggtcagtgaccctagtggtgagcaaaagcagaagctgccaagcagaagaaagaagcagcagaagaggacatttatttcacaggagagagactgtgtctttggcactgattcactcagattgtctgggaaaagactaaaggaacaggaagaaatcagtagcaaaaatcctgctagatcaccagtaactgaaataagaactcaccttttaagtcttaaatctgaacttccagattctccagaaccagttacagaaattaatgaagacagtgtattaattccaccaactgcccaaccagaaaaaggtgttgatacattcctaagaagacctaatttcaccagggcgactacagttcctttacagactctatcagatagcggtagtagtcagcaccttgaacacattcctcctaaaggtagcagtgaacttactactcacgacctaaaaaacattagatttacttcacctgtaagtttggaggcacaaggcaaaaaaatgactgtctctacagataacctccttgtaaataaagctataagtaaaagtggccaactgcccacaagttctaatttagaggcaaatatttcatgttctctaaatgaactcacctacaataacttaccagcaaatgaaaaccaaaacttaaaagaacaaaatcaaacagagaaatctttaaaatctcccagtgacactcttgatggcaggaatgaaaatcttcaggaaagtgagattctaagtcaacctaagagtcttagcctggaagcaacctctcctctttctgcagaaaaacattcttgcacagtgcctgaaggccttctgtttcctgcagaatattatgttagaacaacacgaagcatgtccaattgccagaggaaagtagccgtggaggctgtcattcagagtcatttggatgtcaagaaaaaagggtttaaaaataaaaataaggatgcaagtaaaaatttaaacctttccaatgaggaaactgaccaaagtgaaattaggatgtctggcacatgcacaggacaaccaagttcaagaacctctcagaaacttctctcattaactaaagtcagctctcccgctgggcccactgaagataatgacttgtctaggaaggcagttgcccaagcacctggtagaagatacacaggaaaaagaaaatcagcctgcaccccagcatcagatcattgtgaaccacttttgccaacttctagcctgtcgattgttaacaggtccaaggaagaagtcacctcacacaaatatcagcacgaaaaattatttattcaagtgaaagggaagaaaagtcgtcatcaaaaagaggattccctttcttggagtaatagtgcttatttatccttggatgatgatgctttcacggctccatttcatagggatggaatgctgagtttaaagcaactactgtcttttctcagtatcacagactttcagttacctgatgaagactttggacctcttaagcttgaaaaagtgaagtcctgctcagaaaaaccagtggagccctttgagtcaaaaatgtttggagagagacatcttaaagagggaagctgtatttttccagaggaactgagtcctaaacgcatggatacagaaatggaggacttagaagaggaccttattgttctaccaggaaaatcacatcccaaaaggccaaactcgcaaagccagcatacaaagacgggcctttcttcatccatattactttatactcctttaaatacggttgcgcctgatgataatgacaggcctaccacagacatgtgttcacctgctttccccatcttaggtactactccagcctttggccctcaaggctcctatgaaaaagcatctacagaagttgctggacgaacttgctgcacaccccaacttgctcatttgaaagactcagtctgtcttgccagtgatactaaacaattcgacagttcaggcagcccagcaaaaccacataccaccctgcaagtgtcaggcaggcaaggacaacctacctgtgactgtgactctgtcccgccaggaacacctccacccattgagtcattcacttttaaagaaaatcagctctgtagaaacacatgccaggagctgcataaacattccgtcgaacagactgaaacagcagagcttcctgcttctgatagcataaacccaggcaacctacaattggtttcagagttaaagaatccttcaggttcctgttccgtagatgtgagtgccatgttttgggaaagagccggttgtaaagagccatgtatcataactgcttgcgaagatgtagtttctctttggaaagctctggatgcttggcagtgggaaaaactttatacctggcacttcgcagaggttccagtattacagatagttccagtgcctgatgtgtataatctcgtgtgtgtagctttgggaaatttggaaatcagagagatcagggcattgttttgttcctctgatgatgaaagtgaaaagcaagtactactgaagtctggaaatataaaagctgtgcttggcctgacaaagaggaggctagttagtagcagtgggaccctttctgatcaacaagtagaagtcatgacgtttgcagaagatggaggaggcaaagaaaaccaatttttgatgccccctgaggagactatactaacttttgctgaggtccaagggatgcaagaagctctgcttggtactactattatgaacaacattgttatttggaatttaaaaactggtcaactcctgaaaaagatgcacattgatgattcttaccaagcttcagtctgtcacaaagcctattctgaaatggggcttctctttattgtcctgagtcatccctgtgccaaagagagtgagtcgttgcgaagccctgtgtttcagctcattgtgattaaccctaagacgactctcagcgtgggtgtgatgctgtactgtcttcctccagggcaggctggcaggttcctggaaggtgacgtgaaagatcactgtgcagcagcaatcttgacttctggaacaattgccatttgggacttacttctcggtcagtgtactgccctcctcccacctgtctctgaccaacattggtcttttgtgaaatggtcgggtacagactctcatttgctggctggacaaaaagatggaaatatatttgtataccactattcataa ( SEQIDNO：3 ).
Wild type PALB2 gene encodes 1186 aminoacid altogether, the sequencing data of No. 31 proband shows that the 1709th to 1710 nucleotide of its coding region lacks (disappearance base is AG), there is frameshift mutation, ultimately result in and can only encode 575 aminoacid.
5. use goldstandard (Sanger order-checking) that 5 pathogenic mutation sites are verified
In order to verify the technology accuracy of the present invention, PCR primer is designed in the region at place, 5 mutational sites, after standard PCR amplification, carries out Sanger order-checking, analyze the peak figure situation of target site.The result shows: it is consistent with Sanger sequencing result that the high-flux sequence of the present invention analyzes result, and the Sanger of No. 5, No. 10, No. 15, No. 25 and No. 31 samples checks order gained peak figure respectively as shown in fig. 3 to 7, and in figure, Shadow marks is sudden change place/initiation site.
6. the relevant family members of pair carriers of mutation carry out sudden change checking and family history analysis
Use PCR to combine Sanger sequencing, respectively the family members of the proband that 5 carry breast cancer susceptibility gene mutation carried out the checking in corresponding mutational site, specify mutational site whether produce with disease to be divided into from.The family members participating in checking include maternal and the major families member of paternal (wherein a side exists the related neoplasms family histories such as breast carcinoma).The result shows, the patient with breast cancer shown as in side relatives of family history of breast cancer all carries the mutational site identical with proband, is absent from corresponding mutational site without showing as in side relatives of family history of breast cancer.Result above shows, 5 breast cancer susceptibility gene mutation sites that we find can cause that breast carcinoma occurrence risk raises, and is the pathogenic mutation of hereditary breast cancer.
Catch the associating NGS platform that checks order due to target area chip and there is the feature of the technology such as high flux, high accuracy, high sensitivity so that the time and the cost that detect based on the hereditary tumor susceptibility gene mutation of large-scale crowd are greatly lowered.Meanwhile, set up information analysis method on the platform and follow-up variation is understood and then can simultaneously, comprehensively be detected and annotation hereditary tumor susceptibility gene mutation situation.Therefore, the present invention is not only suitable for the hereditary tumor susceptibility gene mutation detection for carrying out large-scale crowd, substantial amounts of clinical gene variation detection data can be accumulated rapidly simultaneously, provide data support a large amount of, reliable for scientific research, clinical practice and industrialization.
It should be noted that, the detection method of the gene mutation of the hereditary tumor tumor susceptibility gene of the present invention the diagnostic method of non-diseases, because using the sudden change result that the invention detects that only to illustrate that the risk that related individuals suffers from associated cancer is higher, in addition it is also necessary to just to can confirm that Personal situation in conjunction with clinical effectiveness and/or family history analysis etc..
Above content is in conjunction with specific embodiment further description made for the present invention, it is impossible to assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, it is also possible to make some simple deduction or replace.
Claims (12)
1. a hereditary tumor tumor susceptibility gene target sequence catches chip, it is characterized in that, described chip is liquid-phase chip, it is combined with can catch in the hereditary tumor tumor susceptibility gene of 115 shown in table a kind simultaneously at least 5 kinds, preferably at least 10 kinds, preferably at least 20 kinds, preferably at least 30 kinds, preferably at least 50 kinds, preferably at least 80 kinds, preferably at least 100 kinds, preferably at least 110 kinds, preferably all of target acquistion region probe combinations.
2. hereditary tumor tumor susceptibility gene target sequence according to claim 1 catches chip, it is characterised in that described target acquistion region includes all exon regions and exon and intron join domain.
3. hereditary tumor tumor susceptibility gene target sequence according to claim 1 catches chip, it is characterised in that the liquid-phase chip that described liquid-phase chip is is carrier with the NimblegenEZ chip of Roche.
null4. hereditary tumor tumor susceptibility gene target sequence according to claim 1 catches chip，It is characterized in that，Described hereditary tumor is breast carcinoma、Ovarian cancer、Intestinal cancer、Gastric cancer、Carcinoma of prostate、Carcinoma of endometrium、Leukemia、Medulloblastoma、Ganglioneuroblastoma、Neuroblastoma、Multiple Endocrine tumor、Multiple neurofibromatosis、Pulmonary carcinoma、Pulmonary blastoma、Pheochromocytoma、Osteosarcoma、Melanoma、Rhabdomyosarcoma、Basal cell tumor、Parathyroid carcinoma、Thyroid carcinoma、Lymphoma、Endocrine tumor、Skin carcinoma、Smooth cell tumor、Renal carcinoma、Nephroblastoma、Adrenocortical carcinoma、Meningioma、Bladder cancer、Retinocytoma、Chromaffin cell tumor、Glioma、Exophytic osteosarcoma、Gastrointestinal stromal tumor、Thrombocytosis、One or more in cylindroma and cancer of pancreas.
5. a hereditary tumor tumor susceptibility gene target sequence catching method, it is characterised in that described method includes using hereditary tumor tumor susceptibility gene target sequence described in any one of claim 1-4 to catch the step that chip and DNA sample to be captured carry out hybridizing.
6. the detection method of the gene mutation of a hereditary tumor tumor susceptibility gene, it is characterised in that described method includes using hereditary tumor tumor susceptibility gene target sequence described in any one of claim 1-4 to catch the step that chip and DNA sample to be captured carry out hybridizing；With use second filial generation high throughput sequencing technologies to catching the step that the target dna obtained checks order.
7. the detection method of the gene mutation of hereditary tumor tumor susceptibility gene according to claim 6, it is characterised in that described method comprises the steps:
(1) genomic DNA sample to be detected is broken into fragment, it is preferable that be broken into the fragment that length is 220-400bp；
(2) fragment that step (1) is interrupted is purified, end reparation, adds joint, and expands with PCR；
(3) product step (2) obtained and the hereditary tumor tumor susceptibility gene target sequence described in any one of claim 1-4 are caught chip and are hybridized, and capture the DNA fragmentation in described target acquistion region；
(4) DNA fragmentation in described target acquistion region step (3) captured elutes, it is thus achieved that the target dna of needs；
(5) target dna that step (4) obtains is used to build sequencing library；
(6) use the sequencing library that step (5) is obtained by second filial generation high-flux sequence to check order, obtain reads；
(7) reads step (6) obtained compares with reference to genome.
8. the detection method of the gene mutation of hereditary tumor tumor susceptibility gene according to claim 7, it is characterised in that the form of described gene mutation is that base is replaced, inserted or disappearance and fragment deletion or amplification.
9. the detection method of the gene mutation of hereditary tumor tumor susceptibility gene according to claim 7, it is characterised in that the joint in described step (2) is the A joint of CG order-checking platform；Double-stranded DNA, particularly as follows: again carry out PCR, is carried out cyclisation by described (5), carries out enzyme action at 26bp place, described A joint two ends, otch is carried out end reparation, adds B joint, then DNA double chain is separated into strand, and described strand is carried out cyclisation.
10. the detection method of the gene mutation of hereditary tumor tumor susceptibility gene according to claim 7, it is characterized in that, second filial generation high-flux sequence in described step (6) is CG order-checking, and ensure that the original data volume of each described sequencing library reaches more than 0.6Gb, the order-checking degree of depth of target area reaches 400 × more than, target area coverage reaches more than 99%.
11. the detection method of the gene mutation of hereditary tumor tumor susceptibility gene according to claim 7, it is characterised in that described step (7) particularly as follows:
First, reads comparison order-checking obtained, to reference genome, is not allow for inserting and disappearance, it is preferable that compare with Teramap；
Then, identify and be likely to and described with reference to the different region of genome, it would be possible to comparison is picked out to the reads in these regions and carried out local and assemble, and compares assembling the sequence obtained with described reference genome, it is determined that various types of sudden changes；
Thereafter, the sudden change detected is given a mark, it is preferable that give a mark with varScoreVAF and varScoreEAF；
Finally, filtering out lower than predetermined quality, lower than desired depth, preferred alt_depth < 2/allreads < 5, lower than the sudden change of preset frequency, preferred MAF < 0.25, finally suddenlyd change list.
12. use the gene mutation of the hereditary tumor tumor susceptibility gene that the detection method of gene mutation that hereditary tumor tumor susceptibility gene target sequence described in any one of claim 1-4 catches hereditary tumor tumor susceptibility gene described in chip or any one of claim 6-11 detects, described gene mutation is that the coded sequence shown in SEQIDNO:1 lacks the 640th nucleotide A base；Or the coded sequence shown in SEQIDNO:2 is inserted with base A between the 5800th and 5801 nucleotide, or between the 274th and 275 nucleotide, it is inserted with base A, or at the 1219th nucleotide position generation nonsense mutation；Or the coded sequence shown in SEQIDNO:3 lacks at the 1709th to 1710 nucleotide generation base AG.
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| CN106283199A (en) * | 2016-08-27 | 2017-01-04 | 大连晶泰生物技术有限公司 | The capture library of 50 hot spot mutation genes that detection tumor is relevant and test kit |
| CN106319065A (en) * | 2016-09-14 | 2017-01-11 | 埃提斯生物技术（上海）有限公司 | Kit and capture probe for human BRCA1/2 gene detection on basis of high-throughput sequencing |
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| CN111009288A (en) * | 2019-11-28 | 2020-04-14 | 苏州元德基因生物科技有限公司 | Probe design method of CEBPA gene and application thereof |
| CN111009288B (en) * | 2019-11-28 | 2023-08-29 | 苏州元德友勤医学检验所有限公司 | Probe design method of CEBPA gene and application thereof |
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